Biochemical and Molecular Biological Characterization of CAC2, the Arabidopsis thaliana Gene Coding for the Biotin Carboxylase Subunit of the Plastidic Acetyl-Coenzyme A Carboxylase

Abstract

The biotin carboxylase subunit of the heteromeric chloroplastic acetyl-coenzyme A carboxylase (ACCase) of Arabidopsis thaliana is coded by a single gene (CAC2), which is interrupted by 15 introns. The cDNA encodes a deduced protein of 537 amino acids with an apparent N-terminal chloroplast-targeting transit peptide. Antibodies generated to a glutathione S-transferase-CAC2 fusion protein react solely with a 51-kD polypeptide of Arabidopsis; these antibodies also inhibit ACCase activity in extracts of Arabidopsis. The entire CAC2 cDNA sequence was expressed in Escherichia coli and the resulting recombinant biotin carboxylase was enzymatically active in carboxylating free biotin. The catalytic properties of the recombinant biotin carboxylase indicate that the activity of the heteromeric ACCase may be regulated by light-/dark-induced changes in stromal pH. The CAC2 gene is maximally expressed in organs and tissues that are actively synthesizing fatty acids for membrane lipids or oil deposition. The observed expression pattern of CAC2 mirrors that previously reported for the CAC1 gene (J.-K. Choi, F. Yu, E.S. Wurtele, B.J. Nikolau [1995] Plant Physiol 109: 619–625; J. Ke, J.-K Choi, M. Smith, H.T. Horner, B.J. Nikolau, E.S. Wurtele [1997] Plant Physiol 113: 357–365), which codes for the biotin carboxyl carrier subunit of the heteromeric ACCase. This coordination is probably partially established by coordinate transcription of the two genes. This hypothesis is consistent with the finding that the CAC2 and CAC1 gene promoters share a common set of sequence motifs that may be important in guiding the transcription of these genes.

Coordinate Regulation of the Nuclear and Plastidic Genes Coding for the Subunits of the Heteromeric Acetyl-Coenzyme A Carboxylase

“…After transfer of the RNA to nylon membranes (Magna Lift, MSI, Westbourough, MA), hybridizations were conducted in a buffer containing 50% (v/v) formamide at 65°C for 12 to 16 h using 32 P-labeled probes. 32 P-Labeled antisense RNA probes were transcribed from vectors containing the following inserts: the 730 nucleotides at the 3Ј end of the CAC1 cDNA (Choi et al, 1995;; the 195 nucleotides at the 5Ј end of CAC2 cDNA (Sun et al, 1997); the CAC3 (GBGe16) cDNA; and the 1 kb at the 3Ј end of the accD genomic clone. Hybridized membranes were rinsed twice with 2ϫ SSC, 2% (w/v) SDS for 10 min each time at room temperature, and then washed twice with 0.1ϫ SSC, 0.1% (w/v) SDS for 20 min each time at 65°C.…”

“…35 S-Labeled antisense and sense RNA probes were transcribed from vectors containing the following inserts: the 730 nucleotides at the 3Ј end of the CAC1 cDNA (Choi et al, 1995;; the 195 nucleotides at the 5Ј end of CAC2 cDNA (Sun et al, 1997); the CAC3 (GBGe16) cDNA; and the 1 kb at the 3Ј end of the accD genomic clone. After hybridization and washing, the tissue sections were coated with Kodak NTB2 emulsion (Eastman-Kodak, Rochester, NY), exposed for 2 to 4 d, and developed.…”

“…The plastidic heteromeric ACCase consists of four subunits: BCC, BCase, ␣-CT, and ␤-CT. To date, genes for two of these subunits have been characterized from Arabidopsis, the CAC1 gene, which codes for BCC (Choi et al, 1995;, and the CAC2 gene, which codes for BCase (Bao et al, 1997;Sun et al, 1997). In this report, we describe the isolation and characterization of the CAC3 gene (GenBank accession no.…”

A Multisubunit Acetyl Coenzyme A Carboxylase from Soybean1

“…In addition, there is no apparent homology among the sequences immediately upstream or downstream of the cleavage site in BC compared with either ␣-CT or BCCP. Finally, the alignments of the N-terminal sequences corresponding to the transit peptide of BC from soybean, Arabidopsis (Bao et al, 1997;Sun et al, 1997), and tobacco (Shorrosh et al, 1995) demonstrate that they are not well conserved.…”

“…An alternative to protein purification involves the isolation of cDNAs that encode each of the chloroplast ACCase components, and their use in producing the various subunits in vitro. In this regard, DNAs that encode the chloroplast ACCase ␤-CT subunit from pea (Sasaki et al, 1993), BC from tobacco (Shorrosh et al, 1995) and Arabidopsis (Bao et al, 1997;Sun et al, 1997), BCCP from Arabidopsis (Choi et al, 1995) and Brassica spp. (Elborough et al, 1996), and ␣-CT from pea (Pisum sativum) have been reported.…”